Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling

Abstract

Background: During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin.

Methods: Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40).

Results: Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p < 0.001) compared with age- and sex-matched controls.

Conclusions: The EL-NE assay was specific for NE-degraded elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.

Figure 1

Characterization and Specificity of the EL-NE Monoclonal Antibody. (A): Competitive ELISA showing inhibition of free peptide (GGPGFGPGVV), elongated peptide (GGPGFGPGVVG), nonsense peptide (VGAGVPGLGV) and nonsense screening peptide (Biotin-KK- VGAGVPGLGV). (B): EL-NE fragment levels after 48 hours in vitro incubation of intact human elastin separately in buffer (ELN), elastin incubated in the presence of NE, MMP-2, MMP-7, MMP-9 and MMP-12. NE incubated for 48 hours separately in buffer was added as control. Abbreviations: ELN, elastin; NE, Neutrophil elastase; MMP, matrix metalloproteinase.

Figure 2

Cross-Reactivity to Cathepsin. EL-NE fragment levels after 24 hours in vitro incubation of intact human elastin separately in buffer (ELN) and elastin incubated with CatG and NE. EL-NE levels in NE and CatG separately in buffer are also shown. Abbreviations: ELN, elastin; NE, Neutrophil elastase; CatG, Cathepsin G.

Figure 3

Degradation of Elastin in IPF and lung cancer. (A): EL-NE fragment levels in serum from patients with IPF (n = 10) compared with controls (n = 9). ** p < 0.01. (B): EL-NE fragment levels in serum from patients diagnosed with lung cancer (n = 40 in total), SCC (n = 16), adenocarcinoma (n = 16) and SCLC (n = 8) compared with controls (n = 12). **** p < 0.0001. Groups were compared by T-test with Welch correction. Data are shown as the geometric mean (95% CI). Abbreviations: IPF, idiopathic pulmonary fibrosis; SCC, squamous cell carcinoma; SCLC, small cell lung carcinoma.